Restriction Enzymes

Restriction enzyme is a bacterial protein that cleaves DNA at particular locations, these sites are called restricted sites. The restriction enzymes guard against bacteriophages in living bacteria. They identify the bacteriophage and cleave it at its restriction sites, destroying its DNA. Important genetic engineering tools include restriction enzymes. They may be separated from bacteria and applied in research facilities. The recognition sequences, or short and distinct nucleotide sequences, are recognized by restriction enzymes in DNA. When a DNA sequence is recognized by the restriction enzyme, it hydrolyzes the bond between neighboring nucleotides and cleaves the DNA molecule. The bacteria use the enzyme methylases to add the methyl group at the adenine or cytosine bases within the recognition sequence, preventing the DNA sequences from disintegrating.

History 

In the year 1970, the first restriction enzyme was discovered and identified as such. Since then, more than 3000 restriction enzymes have been thoroughly investigated, and more than 600 of them are now commercially available and often employed in labs to modify and alter DNA. In 1963, scientists discovered the two enzymes that limit bacteriophage development in E. coli. One was an enzyme that sliced DNA, whilst the other added methyl groups to DNA. Restriction endonuclease was the name given to it later. These enzymes are divided into Exonucleases and Endonucleases based on how they function.

Exonucleases and Endonucleases

• Exonucleases: Restrictions exonucleases, such as exonuclease I, exonuclease II, etc., are largely responsible for hydrolyzing the terminal nucleotides from the end of DNA or RNA molecules in either a 5' to 3' or 3' to 5' direction.
• Endonuclease: Restrictions endonucleases identify certain base sequences (restricted sites) within DNA or RNA molecules and catalyze the breakdown of internal phosphodiester bonds with enzymes like EcoRI, Hind III, and BamHI.

Recognition Site

DNA molecules contain restriction sites, also known as restriction recognition sites, that are particular (4–8 base pair long) nucleotide sequences that are recognized by restriction enzymes. Because restriction enzymes often bind as homodimers, these sequences are typically palindromic. A particular restriction enzyme may cut the sequence between two nucleotides within its recognition site or somewhere adjacent.

For example, when the palindromic sequence GAATTC is recognized, the common restriction enzyme EcoRI makes a cut between the G and the A on both the top and bottom strands. On each end of AATT, a sticky end—a segment of a DNA strand with no connected complement—remains as a result. A segment of DNA with a complementary overhang can then be ligated into the overhang (another EcoRI-cut piece, for example).

Types

1. Type I enzymes- The DNA at any distant position from a recognition site is cut by these restriction enzymes. The unique ATP and S-adenosyl-L-methionine are what trigger the activation of these restriction enzymes. These enzymes are still unique and possess multiple functions, including methylase and restriction digestion.
2. Type II enzymes- This kind of restriction enzyme can cleave or split DNA from a location close to the actual recognition site. It does need a lot of magnesium to work properly. This enzyme is functional for single use and is independent of methylase.
3. Type III enzymes- Additionally, this restriction enzyme fragments DNA at a location close to the real recognition site. It needs ATP to function, but it doesn't need any hydrolase. The beginning of the reaction requires S-adenosyl-L-methionine. However, once the reaction starts, enzyme activity is irrelevant. With the aid of a modification methylase, this restriction enzyme can aid in the digestion of DNA.
4. Type IV- The type IV restriction enzyme is a unique endonuclease that only works on DNA that has been altered. This restriction endonuclease, which works on DNA, is frequently employed in the biotechnology industry. Methylated and hydroxymethylated enzymes are two examples of restriction enzymes.
5. Type V- The Type V restriction enzyme does not function as a DNA reaction enzyme. An RNA guide known as the gRNAs catalyzes this restriction endonuclease's action on RNA.

Artificial Restriction Enzymes

By linking an artificial nuclease domain to a synthetic DNA-binding domain, restriction enzymes can be created. Such synthetic restriction enzymes can bind to specific DNA sequences and can target big DNA locations (up to 36 bp). The most popular artificial restriction enzymes are zinc finger nucleases, which are typically utilized in genetic engineering but can also be used in more conventional gene cloning procedures. The DNA-binding domain of TAL effectors serves as the foundation for additional synthetic restriction enzymes.

In 2013, the genome editing tool CRISPR-Cas9, based on a prokaryotic virus defense mechanism, was developed. It was quickly embraced in labs. Also created are synthetic ribonucleases that function as RNA restriction enzymes. A PNAzyme is a PNA-based system that mimics ribonucleases for a particular RNA sequence and cleaves at a non-base-paired region (RNA bulge) of the targeted RNA generated when the enzyme binds the RNA. This region is known as the RNA bulge. This enzyme exhibits selectivity by only cleaving at one of two potential cleavage sites that are either kinetically favored or do not have a mismatch.

Nomenclature

There have been numerous identifications of restriction enzymes since their discovery in the 1970s; for instance, more than 3500 distinct Type II restriction enzymes have been characterized. Using a naming scheme based on bacterial genus, species, and strain, each enzyme is named after the bacterium from which it was obtained.
For example, the EcoRI restriction enzyme's name

Importance 

It is crucial to understand restriction enzymes since they are utilized in Restriction Fragment Length Polymorphisms, which reveal genetic differences and mutations, as well as in the treatment of cancer. They are composed of two long strands of DNA fused with these restriction enzymes to locate certain DNA sequences and cleave them there. It is used to distinguish the type of mutation from the variation. Restriction enzymes recognize two nucleotides on one strand of DNA, which then cleave the strand. A specific kind of nucleotide sequence in a segment of DNA can be cut by restriction enzymes. So, DNA analysis is done using these enzymes.

Ways to Study Restriction Enzymes

Here are some of the top methods for researching restriction enzymes:

• Understand the Fundamentals – It is crucial to master the fundamentals of each type of enzyme.
• Experiment - Try to use enzymes in every experiment.
• Imagine- what would occur if the enzyme had not been used in the experiment.
• Try It - Try your hand at writing an essay about the enzyme.

Application

In molecular biology research, restriction endonucleases are frequently employed for the following purposes:

1. Genetic engineering: Restriction endonucleases are most frequently used as a technique in genetic engineering. The host organism's genome can be modified and interesting sequences can be introduced thanks to the endonuclease activity. The host then produces the desired gene product as a result. This idea has numerous biotechnological applications, including the creation of antibiotics, antibodies, enzymes, and several secondary metabolites.
2. Restriction enzymes are used in DNA mapping, sometimes referred to as restriction mapping, to gather structural data on the DNA fragment. This method produces DNA fragments of varied sizes by digesting the DNA with a sequence of restriction enzymes. By using agarose gel electrophoresis to separate the resulting fragments, the separation between the restriction enzyme sites can be calculated. This can be used to ascertain a DNA fragment's structure.
3. Gene sequencing entails digesting a big DNA molecule with restriction enzymes and then running the resultant pieces through a DNA sequencer to determine the nucleotide sequence.
4. Studies on gene expression, mutations, and population polymorphisms are some of the other uses for restriction endonucleases.

Examples

EcoRI, HindIII, and NotI are a few well-known examples of restriction enzymes.

Question 1: Define restriction enzymes and Recognition sites.

Answer:

It is an enzyme that cleaves DNA into pieces at or close to particular molecular recognition sites. A recognition site is a location that a restriction enzyme selects in order to break DNA.
These locations are found on a DNA molecule and contain particular nucleotide sequences (4-8 base pairs)

Question 2: Write the applications of the restriction enzyme.

Answer:

Applications of the restriction enzyme:

1. Genetic engineering
2. Used in methods for DNA fingerprinting.
3. They help in gene cloning, protein expression research, and the insertion of genes into plasmid vectors.
4. By specifically identifying single base variations in DNA as single nucleotide polymorphism, they are also useful to distinguish gene alleles.
5. DNA mapping.
6. Gene sequencing.

Question 3: In which year was the first restriction enzyme identified?

Answer:

 In 1970 the first restriction enzyme was identified.

Question 4: Write the difference between Endonuclease and Exonuclease.

Answer: